The identification of disease causing mutations and the genetic characterization of infectious agents has resulted in the ability to diagnose genetic (including congenital and acquired and infectious) diseases at the molecular level. Molecular diagnostic methods, however, remain time and labor intensive, thus limiting the use and availability of routine molecular testing for patient care. "DNA testing on a chip" (i.e., the use of microchip technology for molecular diagnostics) has recently been touted as the solution to the high cost of molecular-based testing. The design, fabrication processing and use of an integrated microchip based genetic testing system is described. The invention described combines unique designs for several components including a nucleic acid amplifier microchip and separate detector device fabricated onto a silicon substrate and integrated through the use of polymeric materials defining the respective spacing elements, fluid reservoirs and transport channels as well as serving as a packaging material to house the complete unit and its controlling electronic circuitry.
Advances in molecular genetics, derived principally from the human genome project, promise to revolutionize health care in the 21st century. These advances recognize that most human disease is a consequence of variations in the structure of DNA, whether through deleterious mutations or due to a simple difference in the sequence of DNA that predispose to disease. These observations point to the fact that molecular genetic testing is a likely final common pathway to all medical diagnoses. For this reason, one focus in health care, and for biomedical research in the near and long term future will be to develop more advanced systems for molecular genetic based diagnostic testing.
Presently, molecular diagnostic laboratories are in their early stages of evolution. Typically these facilities exist only in large hospitals or academic medical centers, where the service offered focuses on providing a handful of tests for selected disease states. With the advent of the polymerase chain reaction, PCR, a method to amplify a precise fragment of DNA to quantities which can be easily evaluated, there has been a major improvement in the feasibility of performing routine molecular genetic testing. Consequently, PCR has become the primary biochemical technique used in molecular genetic laboratories. Although PCR, and other amplification techniques are highly specific and sensitive, they are still highly labor intensive and consequently very costly.
Thus, despite the importance of molecular genetic testing to improve patient: care, the growth of this discipline is being challenged by the mandate of the health care market to fundamentally reduce the cost of genetic testing. So great are the expectations to contain costs, that given the present state of technology and automation in these laboratories, the promises of molecular genetics for patient care will not be realized because these forms of "esoteric" testing will be unaffordable.
Several factors contribute to the high cost clinical molecular genetic testing. Although microchip technology is being developed for several specific applications in the molecular genetic research laboratory, the utility of this technology for the purpose of testing has not been realized. Introduction of this technology into the clinical laboratory will dramatically decrease the cost and labor associated with molecular diagnostics, thereby increasing the availability and potential clinical applications for genetic testing. How molecular genetic testing is presently being performed, and the opportunities to improve it based on microelectromechanical (MEMs) technology is being evaluated by several research groups throughout the world.
However, based on the current methods of performing molecular genetic testing and the costs associated with each operational step, the most significant costs are the so-called front-end which include specimen procurement and nucleic acid extraction. Specifically, collection of blood from the patient is an invasive procedure which requires a trained medical technician. Large specimen sizes are convenient for manual processing, but necessitate large scale nucleic acid extractions which use costly reagents. Although first generation automated DNA extractors have been available, these instruments use large quantities of toxic chemicals and are not applicable to small specimens. Similarly, the test set-up, namely the assembly of the chemical reactions involved in the DNA amplification procedure, are typically done manually. Only recently have first generation robotics systems been commercialized, which are predicted to reduce the cost of labor and may also eliminate errors and increase throughput.
Of equal importance to the labor costs, however, are the costs of the reagent used in DNA amplification based chemistry. In this regard, reduction to a microscale reaction volume, such as that conceived with a microfabricated version of a thermal controlled DNA amplifier would have overall a significant impact on the reduction of the cost of genetic testing.
One solution to the problem of sample procurement and DNA extraction is the replacement of DNA in solution to one where it is enmeshed in a solid support system, such a paper. The use of FTA.TM. coated paper (a product of Fitzco, Inc. of Minneapolis, Minn.) for blood stains and other tissue sources works in this manner. In extensive comparative analyses with conventional DNA extractions, DNA extractions on FTA.TM. paper have demonstrated significant ease in use and reduced cost in performing routine clinical molecular genetic testing. Phased efforts to use the paper-based FTA in the form of a genetic test collection kit are underway, but no such efforts are directed toward a microchip application of this material.
Exploitation of silicon as a substrate for micromachined devices is well established in the engineering fields. Microelectromechanical systems (MEMS) refers to the output of microfabricated devices including those for uses ranging from automotive parts to the airline industry. MEMS have a particular usefulness in biological applications due to their requirement for small sample sizes, low energy, and nominal forces. The increased efficiency of MEMS-based instruments, however, has yet to be realized commercially in biomedical applications, where the need for economy in manufacture, ease of operation, reduction of consumables and the mobilization of the laboratory operation to point-of-care testing are evident. While the future looks promising for the continued development of MEMS for biomedical applications, especially for the clinical chemistry, relatively little research has been applied to the field of molecular genetics utilizing MEMS technology.
Development of chip based technologies for testing DNA has focused primarily in two areas: one on a miniaturized thermal cycling device, and the second on a variety of chip based detection methods. To date, however, no successful integration of these components along with microfluidic controls, on chip electronics and attempts at small and portable controlling mechanism(s) have been achieved.
Considerable work in the area of a microchip based thermocycling device has been described. The initial research was based on the use of a simple heater chip held in a block type apparatus which clamped the chip to a plastic or metal reservoir. The microchips themselves have been fabricated from glass or combinations of silicon bonded to glass by anodic or silicone rubber fixation. Amplification of DNA or RNA based templates has been achieved using on-chip polysilicon thin film heaters, or externally by means of a Peltier heater-cooler. Moreover, in these cases, nucleic acid amplification was accomplished using the polymerase chain reaction, ligase chain reaction and even isothermal enzyme based amplifications.
Using chip-based devices, there has been research into what combination of materials are compatible with the enzyme-based reactions. In general, standard materials common to the silicon processing industry have been explored including thermal deposition of silicon oxide. Preliminary work has also shown how conventional plastics such as polyethylene and polypropylene can be used to "passivate" the silicon chip surface. To date, however, no work is known that outlines the use of dipped or coated polymeric materials on processed silicon as a means to facilitate the amplification chemistry.
Work on chip based detectors is more established with most efforts focused on the use of fluorescent based optical material. Early research by Fedor et. al. established that silicon could serve as a substrate onto which organic molecules such as DNA could be synthesized. The process, now commercialized by Affymetrix Inc. of Santa Clara, Calif., involves the use of serial photolithographic steps to build oligonucleotides in situ at a specific addressable position on the chip. The strategy of addressing specific nucleic acid sequences synthesized off-chip, then hybridized to a particular location on a chip by electrical attraction to a charged microelectrode has been developed by Nanogen Inc. Variation on the theme of microaddressable arrays has recently led to the evaluation of chips for sequence analysis of uncharacterized genetic material, mutational analysis of a known gene locus, and for the evaluation of a particular cell or tissue's profile of gene expression for the whole complement of the human DNA sequence. However, in each case, these methodologies rely on the use of laser activated fluorescence of addressable signals on a microchip.
Another approach to micromachined based detection systems involves the use of capillary electrophoresis. In this case, MEMS-based processing of silicon, glass and plastic has been employed to create microchannels, capillaries and reservoirs in which samples of fluid containing large molecular weight as well as small sized, amplified DNA fragments which are electrophoresed in the presence of an applied voltage. This strategy has been commercialized by companies such as Soane and Caliper Technologies Inc. Recently, Cephiad Inc. has adopted a similar approach to combine a microchip based thermocycler with capillary electrophoresis in a single unit. Such devices have demonstrated high resolution of DNA fragments of molecular weights ranging from 60-2000 base pairs in time superior to macroscale capillary electrophoresis instruments. In all cases, the performance of the various detection systems is comparable, and in many cases better than that of conventional agarose and polyacrylamide gel electrophoresis. However, no experience of the use of a MEMS-based device which employs transduction of a mechanical based signal to an electronic readout has been disclosed.